Martin Huisman

Impaired immune responses and antigen-specific memory CD4+ T cells in hemodialysis patients

Serologic responses to T cell-dependent vaccinations are severely attenuated in patients with ESRD, but the reasons for this is unknown. In this study, a detailed analysis of antigen-specific T cell responses was performed. Patients on hemodialysis and age- and gender-matched healthy control subjects were vaccinated with hepatitis B surface antigen (HBsAg), antigen-specific CD4(+) T cells were monitored at regular intervals with intracellular cytokine staining and proliferation assays. IL-2-and IFN-gamma-producing CD4(+) T cells were identified as either central or effector memory CD4(+) T cells using antibodies directed against CD45RO and the chemokine receptor CCR7. Control subjects mounted a memory T cell response comprising both central and effector memory CD4(+) T cells, with the central memory response occurring 1 wk before the effector memory response. IL-2(+) HBsAg-specific memory CD4(+) T cells were primarily detected within the effector population. Patients with ESRD showed a delayed response of IL-2-and IFN-gamma-producing central memory CD4(+) T cells, but their maximal responses were similar to those of control subjects. In contrast, patients with ESRD produced only 6.3% of the IL-2(+) HBsAg-specific effector memory CD4(+) T cells produced by control subjects (0.5 +/- 0.2 x 10(4)/L versus 8 +/- 3.5 x 10(4)/L; P < 0.001), and this impaired response correlated with antigen-specific T cell proliferation and anti-HBsAg IgG titers. In conclusion, the production of antigen-specific effector memory CD4(+) T cells after vaccination, which is critical to achieve an adequate humoral response, is severely impaired in patients with ESRD.

Unacylated Ghrelin Rapidly Modulates Lipogenic and Insulin Signaling Pathway Gene Expression in Metabolically Active Tissues of GHSR Deleted Mice

Background There is increasing evidence that unacylated ghrelin (UAG) improves insulin sensitivity and glucose homeostasis; however, the mechanism for this activity is not fully understood since a UAG receptor has not been discovered. Methodology/Principal Findings To assess potential mechanisms of UAG action in vivo, we examined rapid effects of UAG on genome-wide expression patterns in fat, muscle and liver of growth hormone secretagogue receptor (GHSR)-ablated mice using microarrays. Expression data were analyzed using Ingenuity Pathways Analysis and Gene Set Enrichment Analysis. Regulation of subsets of these genes was verified by quantitative PCR in an independent experiment. UAG acutely regulated clusters of genes involved in glucose and lipid metabolism in all three tissues, consistent with enhancement of insulin sensitivity. Conclusions/Significance Fat, muscle and liver are central to the control of lipid and glucose homeostasis. UAG rapidly modulates the expression of metabolically important genes in these tissues in GHSR-deleted mice indicating a direct, GHSR-independent, action of UAG to improve insulin sensitivity and metabolic profile.

Expansion of cytolytic CD4+CD28− T cells in end-stage renal disease

Cytomegalovirus (CMV) seropositivity is associated with increased risk for atherosclerotic disease in patients with end-stage renal disease. This association is due to a unique peripheral blood CD4(+) T cell population which lack CD28 (CD4(+)CD28(-) T cells). Here we found that this patient population has a significant age-dependent increase of CD4(+)CD28(-) T cells that comprise over half of the circulating CD4 T cells in some. Patients over 50 years of age have a 50-fold higher percentage of CD4(+)CD28(-) T cells compared to seronegative patients and a 5-fold higher percentage when compared to seropositive healthy controls. Stimulation by CMV-antigen or by polyclonal stimulation using PMA and ionomycin showed that CD4(+)CD28(-) cells in patients with end stage renal disease degranulated and secreted interferon gamma thus indicating that they are cytolytic. The average anti-CMV IgG titer displayed a remarkable age-dependent increase only in patients with end stage renal disease. These findings are highly suggestive of repetitive antigenic stimulation of the immune system in patients with end stage disease by subclinical CMV reactivation which might contribute to increased atherosclerotic risk.

Des-acyl ghrelin analogs prevent high-fat-diet-induced dysregulation of glucose homeostasis

There is clinical evidence that des‐acyl ghrelin (DAG) favorably modulates glucose and lipid metabolism, although its mode of action is unknown. A murine model of prediabetes was used to assess possible mechanisms of action for DAG and a newly developed bioactive analog, AZP531. C57BL/6J mice were infused with saline, DAG, or AZP531 continuously for 4 wk, and fed either normal diet (ND) or normal diet for 2 wk followed by a high‐fat diet (HFD) for 2 wk. Compared with mice in the ND group, HFD increased body and fat mass, caused glucose intolerance and insulin resistance, had proinflammatory effects in white adipose tissue, and caused lipid accumulation in brown adipose tissue. DAG and AZP531 treatment prevented HFD‐induced proinflammatory effects, stimulated expression of mitochondrial function markers in brown adipose tissue, and prevented development of a prediabetic metabolic state. AZP531 also prevented a HFD‐induced increase in acyl ghrelin levels. Our data indicate DAG analogs as potential treatment for the prevention of metabolic syndrome.—Delhanty, P. J. D., Huisman, M., Baldeon‐Rojas, L.Y., van den Berge, I., Grefhorst, A., Abribat, T., Leenen, P. J. M., Themmen, A. P. N., van der Lely, A.‐J. Des‐acyl ghrelin analogs prevent high‐fat diet‐induced dysregulation of glucose homeostasis. FASEB J. 27, 1690–1700 (2013). www.fasebj.org

IL-2 Producing Memory CD4+ T Lymphocytes Are Closely Associated with the Generation of IgG-Secreting Plasma Cells

The role of specific CD4+ T cell subsets in the induction of humoral immune responses in humans is largely unknown. In this study, the generation of hepatitis B surface Ag-specific CD4+ T lymphocytes following vaccination was closely monitored and characterized at the single-cell level. The appearance and absolute numbers of hepatitis B surface Ag-specific IL-2 producing effector memory CD4+ T lymphocytes was solely and tightly related to Ab titers reached. This relation remained present many years after vaccination. Subsequently, a relation was found between Ab titers and number of IL-2 producing memory CD4+ T lymphocytes for various other Ags. These observations matched the findings of an in vitro assay, using different T cell subsets to induce B cell differentiation into IgG-producing plasma cells. By depleting for IL-2 producing memory T cells, we demonstrated that these cells are important for B cell differentiation into IgG-producing plasma cells. Finally, blocking the action of IL-2 with an IL-2R-α Ab inhibited the differentiation of B lymphocytes into IgG-producing plasma cells. Based on these findings, we conclude that the development of Ag-specific IL-2-producing memory T cells appears to be essential for the development of IgG-secreting plasma cells in humans.

Does des-acyl ghrelin improve glycemic control in obese diabetic subjects by decreasing acylated ghrelin levels?

OBJECTIVE The objective of this study was to assess the effects of a continuous overnight infusion of des-acyl ghrelin (DAG) on acylated ghrelin (AG) levels and glucose and insulin responses to a standard breakfast meal (SBM) in eight overweight patients with type 2 diabetes. Furthermore, in the same patients and two additional subjects, the effects of DAG infusion on AG concentrations and insulin sensitivity during a hyperinsulinemic-euglycemic clamp (HEC) were assessed. RESEARCH DESIGN AND METHODS A double-blind, placebo-controlled cross-over study design was implemented, using overnight continuous infusions of 3 and 10  μg DAG/kg per h and placebo to study the effects on a SBM. During a HEC, we studied the insulin sensitivity. RESULTS We observed that, compared with placebo, overnight DAG administration significantly decreased postprandial glucose levels, both during continuous glucose monitoring and at peak serum glucose levels. The degree of improvement in glycemia was correlated with baseline plasma AG concentrations. Concurrently, DAG infusion significantly decreased fasting and postprandial AG levels. During the HEC, 2.5  h of DAG infusion markedly decreased AG levels, and the M-index, a measure of insulin sensitivity, was significantly improved in the six subjects in whom we were able to attain steady-state euglycemia. DAG administration was not accompanied by many side effects when compared with placebo. CONCLUSIONS DAG administration improves glycemic control in obese subjects with type 2 diabetes through the suppression of AG levels. DAG is a good candidate for the development of compounds in the treatment of metabolic disorders or other conditions with a disturbed AG:DAG ratio, such as type 2 diabetes mellitus or Prader-Willi syndrome.

The acylated (AG) to unacylated (UAG) ghrelin ratio in esterase inhibitor‐treated blood is higher than previously described

The acylated/unacylated ghrelin (AG/UAG) ratio has been reported to range from 0·02 to 0·3, suggesting biologically relevant independent regulation of each ghrelin isoform. However, AG is deacylated to UAG by esterases in blood samples, and esterase inhibition is critical for their accurate measurement. Our hypothesis is that at least part of the variation in reported AG and UAG values is due to inconsistent sample preparation.

Amelioration of renal ischaemia-reperfusion injury by synthetic oligopeptides related to human chorionic gonadotropin

BACKGROUND We have previously reported that small synthetic oligopeptides related to human beta-chorionic gonadotropin (beta-hCG) can reduce inflammation. Here we investigated whether such oligopeptides can reduce renal ischaemia-reperfusion injury in the mouse. METHODS Ten different oligopeptides were administered 1 min before induction of renal ischaemia and 1 min before reperfusion. RESULTS Survival at 72 h post-reperfusion was significantly higher in mice treated with oligopeptides MTRV, LQG, VLPALPQ or AQGV as compared to placebo-treated mice. Some oligopeptides were more effective than others. AQGV completely prevented mortality and best preserved kidney function. Next, AQGV was tested in a dose-escalating study in a range of 0.3-30 mg/kg. A survival gain was observed with all doses. Improvement of kidney function was observed from 1 mg/kg. Highest survival and best preserved kidney function were observed at 3 and 10 mg/kg. Upon treatment with AQGV, a significantly lower influx of neutrophils was found, apoptosis was decreased, whereas tubular epithelial cell proliferation was significantly increased at 24 h post-reperfusion. Serum levels of TNF-alpha, INF-gamma, IL-6 and IL-10 were significantly decreased at 24 h post-reperfusion. E-selectin mRNA levels in kidneys were significantly decreased at 6 h post-reperfusion. AQGV did not reduce mortality when treatment was started after reperfusion. CONCLUSIONS This study shows that small oligopeptides related to the primary structure of beta-hCG, especially AQGV, are promising potential drugs for preventing the development of renal ischaemia-reperfusion injury.

Development of potent selective competitive-antagonists of the melanocortin type 2 receptor

Cushings disease, a hypercortisolemic state induced by an ACTH overexpressing pituitary adenoma, causes increased morbidity and mortality. Selective antagonism of the melanocortin type 2 receptor (MC2R) may be a novel treatment modality. Five structurally related peptides with modified HFRW sites but intact putative MC2R binding sites were tested for antagonistic activity at MC1R, MC2R/MRAP, MC3R, MC4R and MC5R. Two of these peptides (GPS1573 and GPS1574) dose-dependently antagonized ACTH-stimulated MC2R activity (IC50s of 66±23 nM and 260±1 nM, respectively). GPS1573 and 1574 suppressed the Rmax but not EC50 of ACTH on MC2R, indicating non-competitive antagonism. These peptides did not antagonize α-MSH stimulation of MC1R and antagonized MC3, 4 and 5R at markedly lower potency. GP1573 and GPS1574 antagonize MC4R with IC50s of 950 nM and 3.7 μM, respectively. In conclusion, two peptide antagonists were developed with selectivity for MC2R, forming a platform for development of a medical treatment for Cushings disease.

Unsaturated fatty acids prevent desensitization of the human growth hormone secretagogue receptor by blocking its internalization

The composition of the plasma membrane affects the responsiveness of cells to metabolically important hormones such as insulin and vasoactive intestinal peptide. Ghrelin is a metabolically regulated hormone that activates the G protein-coupled receptor GH secretagogue receptor type 1a (GHSR) not only in the pituitary gland but also in peripheral tissues such as the pancreas, stomach, and T cells in the circulation. We have investigated the effects of lipids and altered plasma membrane composition on GHSR activation. Oligounsaturated fatty acids (OFAs) disrupt the structure of membranes and make them more fluid. Prolonged (96 h), but not acute, treatment of the GHSR cells with the 18C OFAs oleic and linoleic acid caused a significant increase in sensitivity of the receptor to ghrelin (EC(50) reduced by a factor of 2.4 and 2.9 at 60 and 120 microM OFAs, respectively). OFAs were found to block the inhibitory effects of ghrelin pretreatment on subsequent ghrelin responsiveness, suggesting that OFAs suppress desensitization of GHSR. Radioligand displacement studies did not show a significant shift in receptor binding after incubation with OFAs. However, it was found that OFA treatment suppressed GHSR internalization, likely explaining OFA-induced refractoriness to ligand-induced desensitization. The involvement of lipid rafts in this process was indicated by the altered responsiveness of GHSR under conditions that alter membrane cholesterol. In conclusion, our findings demonstrate the importance of membrane composition for GHSR activation and desensitization and indicate at least part of the mechanism through which OFAs and cholesterol could affect ghrelins activity in vivo.